(vertebrate evolutionyAmniotayChondrichthyesystarry skateymolecular dating)

The Chondrichthyes (cartilaginous fishes) are commonly accepted as being sister group to the other extant Gnathostomata (jawed vertebrates). To clarify gnathostome relationships and to aid in resolving and dating the major piscine divergences, we have sequenced the complete mtDNA of the starry skate and have included it in phylogenetic analysis along with three squalomorph chondrichthyans—the common dogfish, the spiny dogfish, and the star spotted dogfish—and a number of bony fishes and amniotes. The direction of evolution within the gnathostome tree was established by rooting it with the most closely related nongnathostome outgroup, the sea lamprey, as well as with some more distantly related taxa. The analyses placed the chondrichthyans in a terminal position in the piscine tree. These findings, which also suggest that the origin of the amniote lineage is older than the age of the oldest extant bony fishes (the lungfishes), challenge the evolutionary direction of several morphological characters that have been used in reconstructing gnathostome relationships. Applying as a calibration point the age of the oldest lungfish fossils, 400 million years, the molecular estimate placed the squalomorphy batomorph divergence at '190 million years before present. This dating is consistent with the occurrence of the earliest batomorph (skates and rays) fossils in the paleontological record. The split between gnathostome fishes and the amniote lineage was dated at '420 million years before present. The relationship between gnathostomous fishes and their terrestrial relatives is of fundamental importance for the understanding of vertebrate evolution. Molecular analyses of this relationship have addressed in particular the question of whether, among extant fishes, the lungfishes or the coelacanth are the sister group to terrestrial vertebrates. However, although these analyses have differed with respect to the taxa included, a teleostean (1–4) or chondrichthyan (5) rooting of the gnathostome tree has been a common characteristic, and these studies have, in general, supported a sister group relationship between lungfishes and amniotes (or tetrapods). Because the application of rooting automatically gives evolutionary direction to a tree, it is essential that rooting is performed by using an outgroup that is unambiguously positioned without the ingroup taxa. The commonly applied teleostean rooting of the vertebrate tree is incompatible with piscine paleontology (6, 7) whereas the chondrichthyan rooting is subjective in the sense that it assumes a priori that chondrichthyans are the sister group of all other extant gnathostomes. Therefore, the application of either the teleostean or chondrichthyan rooting is inconsistent with the criterion that unequivocal outgroups should be used to establish the polarity of phylogenetic trees. The conclusions based on the teleostean and chondrichthyan rooting have been challenged in two recent molecular studies (8, 9) in which the gnathostome tree was rooted by using non-gnathostome taxa. The first study indicated that the lungfishes have a basal position in the piscine tree and that the separation between extant bony fishes and amniotes preceded the divergence of the extant bony fishes. The second study refuted the commonly held belief that the Chondrichthyes are basal to other gnathostomes. This analysis, with only the spiny dogfish, Squalus acanthias, representing squalomorph chondrichthyans, did not, however, resolve the relationship between the coelacanth, the chondrichthyans, and the teleosts. To examine this relationship in greater detail, we have, in the present study, broken up the chondrichthyan branch by including mitochondrial genes of three other chondrichthyans, the common dogfish, Scyliorhinus canicula, (10), the star spotted dogfish, Mustelus manazo, (11), and the starry skate, Raja radiata, (present study). Thus, the chondrichthyans are represented by a total of three squalomorphs and one batomorph. The divergence between squalomorphs (sharks) and batomorphs (skates and rays) is paleontologically dated to the early Jurassic (6, 7, 12). Even though this is the minimum age for the squalomorphybatoid divergence, the inclusion of the skate, in addition to strengthening the phylogenetic analysis, makes it possible to test a molecular estimate of the divergence time between the squalomorphs and the skate against the paleontological record of the Batomorphii. MATERIALS AND METHODS Enriched mtDNA was isolated from frozen liver of the starry skate, Raja radiata, following described procedures (13). The specimen was collected in Faxafloi, Iceland, by Oskar Gudmundsson. The mtDNA was digested separately with BlnI and BclI. Digested DNA fragments were separated on an agarose gel and were excised, electroeluted, and ligated. With the exception of parts of the NADH2 and NADH5 genes, which were PCR-amplified and direct-sequenced, natural clones covered the whole molecule. The mtDNA of the starry skate has been deposited in the GenBank database with accession number AF106038. Users of the sequence are kindly requested to refer to the present paper and not only to the accession number of the sequence. The phylogenetic analyses included all published piscine mtDNAs together with a comprehensive selection of taxa represented by complete mtDNAs, namely sea lamprey, Petromyzon marinus (14); African lungfish, Protopterus dolloi (3); The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: MYBP, million years before present; ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor joining; NADH1-6, subunits 1–6 of nicotinamide adenine dinucleotide dehydrogenase. Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF106038). *To whom reprint requests should be addressed. e-mail: ulfur. [email protected].